INTEGRATED DNA GENOTYPING AND SEQUENCING MICROSYSTEMS

DNA基因分型和测序的分析方法被连接到从DNA来源材料开始并提取遗传信息的多步骤处理系统中。在传统方法中，处理系统处理一批微升体积的反应样品，以及匹配的液体处理设备和电泳分析设备。项目1将提供一种系统级别的方法来评估计划项目中开发的集成纳米升体积组件和制造技术。微加工部件的成功将通过它们作为一个自主的多步骤处理系统工作的能力来衡量。项目1有四个具体目标：1)开发集成纳升规模DNA分析所需的微流体。微流控部件将被用来构建一个可以执行和监控多个平行反应的集成系统。还将解决与微升体积源样品的接口问题。2)在一个集成系统中演示测序和基因分型反应。微流控设备将进行优化，以用于桑格测序或基因组扩增反应和变性凝胶电泳。系统级设备将执行和监测聚合酶反应、反应后处理和电泳带迁移，只需最少的操作员干预。3)在集成系统中展示高质量的基因组DNA测序。将提高集成纳升体积系统的分辨率，以匹配现有微升规模的集成纳升体积系统的水平。将改进集成纳升体积系统，以匹配现有微升规模的技术。这将包括改进的荧光探测器(项目3)和更高分辨率的凝胶(项目2)。这一目的的成功完成将导致一种装置，该装置可以从模板DNA样本中获得500-1000bp的序列。4)在集成设备中演示复杂的排序策略。从单个DNA样本中获得最大信息的复杂测序策略，如双向测序或同时限制性内切酶消化，将被添加到集成测序设备中。还将审查质量控制、准确性和数据重复性等问题。

Analysis methods for DNA genotyping and sequencing are linked into multiple-step processing system that begin with DNA source material and extract genetic information. In conventional methods, the processing system operates on batches of microliter-volume reaction samples, together with matched-liquid handling devices and electrophoretic analysis equipment. Project 1 will provide a systems-level approach to evaluate the integrated nano-liter volume components and fabrication technologies developed in the Program Project. The success of microfabricated components will be measured by their ability to work as an autonomous multi-step processing system. Project 1 has four Specific Aims: 1) Develop the microfluidics necessary for integrated nanoliter-scale DNA analysis. Microfluidic components will be used to construct an integrated system that can perform and monitor multiple parallel reactions. An interface with microliter-volume source samples will also be addressed. 2) Demonstrate sequencing and genotyping reactions in an integrated system. Microfluidic devices will be optimized for use with Sanger sequencing or genomic amplification reactions and denaturing gel electrophoresis. The system-level device will perform and monitor polymerase reactions, post reaction treatment, and electrophoretic band migration, with minimal operator intervention. 3) Demonstrate high-quality genomic DNA sequencing in an integrated systems. The resolution of the integrated nanoliter-volume system will be improved to match the level of existing microliter-scale integrated nanoliter-volume system will be improved to match the level of existing microliter-scale technologies. This will include improved fluorescence detectors (with Project 3) and higher resolution gels (with Project 2). Successful completion of this aim will result in a device that can obtain 500-1000 bp of sequence from a sample of template DNA. 4) Demonstrate complex sequencing strategies in an integrated device. Complex sequencing strategies that obtain maximal information from a single DNA sample, such as bi-directional sequencing or simultaneous restriction digestion, will be added to the integrated sequencing device. Issues of quality control, accuracy, and data reproducibility will also be examined.